ABSTRACT

Ocular glands play a critical role in eye health through the secretion of factors directly onto the ocular surface. The cornea is a normally transparent tissue necessary for visual acuity located in the anterior segment of the eye. Corneal damage can occur during microbial infection of the cornea, resulting in potentially permanent visual deficits. The involvement of ocular glands during corneal infection has been only briefly described. We hypothesized that ocular glands contribute to resistance as an arm of the eye-associated lymphoid tissue and may also be susceptible to infection secondary to microbial keratitis. Utilizing a mouse model of herpes simplex virus 1 (HSV-1) keratitis, we found that infection of corneas resulted in subsequent infection of ocular glands, including harderian glands (HGs) and extraorbital glands. Similarly, infection of corneas with Pseudomonas aeruginosa resulted in secondary infection of ocular glands. A robust immune response, characterized by increased numbers of immune cells and inflammatory mediators, occurred within ocular glands following HSV-1 keratitis. Removal of HGs altered corneal resistance to HSV-1, as measured by increased viral load, decreased corneal edema, and decreased inflammatory cell infiltration. These novel findings suggest that ocular glands are involved in microbial keratitis through their susceptibility to secondary infection and contribution to corneal resistance.

IMPORTANCE Microbial keratitis accounts for up to 700,000 clinical visits annually in the United States. The involvement of ocular glands during microbial keratitis is not readily appreciated, and treatment options do not address the consequences of ocular gland dysfunction. The present study shows that ocular glands are susceptible to direct infection by common ocular pathogens, including HSV-1 and Pseudomonas aeruginosa, subsequent to microbial keratitis. Additionally, ocular glands contribute soluble factors that play a role in corneal resistance to HSV-1 and alter viral load, corneal edema, and immune cell infiltration. Further studies are needed to elucidate the mechanisms by which this occurs.

INTRODUCTION

Ocular glands are vital in the maintenance of eye health (1). These glands secrete a variety of factors onto the surface of the eye that constitute the tear film, including antimicrobial factors, neuropeptides, mucins, lipids, and nutrients (1, 2). Ocular glands are considered eye-associated lymphoid tissue capable of supporting lymphoid follicle formation, immune cell infiltration, and expansion of lymphocytes, as reported in Sjögren’s syndrome and dry eye models (2–4). In addition, ocular glands are a constitutive source of IgA-secreting plasma cells, complement, lysozyme, and other protective factors within glands (1, 5). These glands access the ocular surface via valveless ducts that allow for the passage of glandular products onto the corneal surface (6). Secretion is regulated by the release of neurotransmitters from parasympathetic and sympathetic nerves and can be upregulated in response to irritation of the cornea (7). In humans, each eye has a designated lacrimal gland located in the superior retro-orbital space lateral to the eye (8). Mice have three lacrimal glands: harderian glands (HGs), extraorbital lacrimal glands (ELGs), and intraorbital lacrimal glands (ILGs) (9). While murine lacrimal glands overlap in structure and function with each other, as well as with human lacrimal glands, a variety of differences exist between them (10). HGs, but not ELGs or ILGs, produce porphyrin, which is thought to be involved in the thermoregulation of mice. HGs can also produce murine pheromones. HGs are lacrimal glands connected to a nictitating membrane found in many rodents, birds, and reptiles but not in humans (10, 11).

Glandular products constituting the tear film help protect the structure and function of the cornea (1). The cornea is a normally transparent tissue in the anterior segment of the eye and plays a critical role in focusing light onto the retina for vision (12). Corneal transparency is due, in part, to its avascular, immune privileged status and the lamellar architecture of collagen in the stroma that prevents light scatter (12, 13). The avascular nature of the cornea reduces efficient transport of oxygen and nutrients uniformly from within; however, the tear film disperses necessary components across the epithelial surface, where these components can diffuse into the tissue (13).

In 2010, the Centers for Disease Control and Prevention reported that 700,000 clinical visits occurred due to microbial infections of the cornea, or microbial keratitis, in the United States (14). Depending on the pathogen, tissue pathology can occur by direct cytopathic effects (e.g., virus), secreted toxins (e.g., bacteria), or immune-mediated damage that can result in opacity and scar formation of the cornea (15, 16). Host clearance of the insulting pathogen can also lead to corneal neovascularization (comprised of blood and lymphatic vessel formation), as in cases of herpes simplex virus 1 (HSV-1) keratitis (15). In viral and bacterial keratitis models, there is a robust innate immune response involving the infiltration of neutrophils into the cornea (17–21). While neutrophils produce a variety of antimicrobial factors, they are also highly inflammatory and susceptible to HSV-1 infection (17, 21).

The potential susceptibility of ocular glands to pathogens has been acknowledged (22–28). Due to the anatomical connection between oculars glands and the ocular surface, we hypothesized that these glands are susceptible to direct infection by pathogens subsequent to microbial keratitis. Since ocular glands are part of the eye-associated lymphoid tissue, we also hypothesized that these glands contribute to corneal resistance to infection. To test the predicted susceptibility of HGs and ELGs to secondary infection following microbial keratitis, we used a well-characterized ocular HSV-1 mouse model. In a time-dependent fashion, HGs and ELGs were infected with HSV-1 subsequent to corneal infection. The susceptibility of these glands was not restricted to a viral pathogen, as Pseudomonas aeruginosa also infected HGs and ELGs secondarily to corneal infection. HSV-1 infection of ocular glands correlated with a robust immune response, including increased numbers of immune cells and inflammatory mediators. Removal of HGs altered the susceptibly of corneas to HSV-1 infection, as measured by increased viral load, decreased corneal edema, and decreased inflammatory cell infiltration. Taken together, these novel findings suggest that microbial infection of the cornea can result in ocular gland infection and, conversely, that these glands can contribute to corneal resistance to infection.

RESULTS

HSV-1 and P. aeruginosa infect ocular glands secondarily to corneal infection.Experimental HSV-1 infection of the cornea can lead to infection of secondary tissues not routinely evaluated for infection, such as the olfactory bulb (29). To establish whether ocular glands are also susceptible to infection subsequent to HSV-1 keratitis, the corneas of CD-1 mice were scarified and topically infected, and ocular glands, including HGs and ELGs, were harvested at days 3, 5, and 7 postinfection (p.i.) and assayed for infectious virus. HSV-1 was detected in HGs by day 3 p.i., whereas virus was not detected until day 5 p.i. in ELGs by plaque assay (Fig. 1A). Significantly more infectious virus was detectable in both glands at day 7 p.i., suggesting that active viral replication had taken place within them.

Whereas the plaque assay is useful for the detection of infectious virus, it is inherently less sensitive than other assays for the detection of virus, viral genes, or virus-encoded proteins (29). Therefore, to determine if HSV-1 trafficked to the HGs earlier than day 3 p.i., expression of the HSV-1 early β lytic gene, the thymidine kinase gene, was assessed in the tissue 1 day p.i. While there was no detection of HSV-1 thymidine kinase mRNA in uninfected HGs, it was detected within HGs at 1 day p.i. (Fig. 1B).

We reasoned that HSV-1 may travel from corneas to HGs and ELGs through the valveless ducts that connect the glands to the ocular surface (1). Therefore, to determine if virus applied to the ocular surface might enter HGs in this manner, Evans blue dye was topically added to the ocular surface following corneal scarification, and HGs were removed 4 h later. The dye was present in the HGs at this time, as determined by dark blue staining of the normally pink glands (Fig. 1C).

To determine whether the susceptibility of ocular glands to pathogen entry was unique to small, viral pathogens, like HSV-1, we utilized P. aeruginosa, a larger bacterial pathogen, for corneal infection. Following corneal scarification and topical inoculation of CD-1 mice, bacteria were detected within ocular glands at days 1 and 3 p.i. (Fig. 1D). As observed with HSV-1, CFU counts of P. aeruginosa were detected at higher numbers and earlier in HGs than in ELGs. We interpret these results to suggest that ocular surface contents have access to ocular glands and that this may be one mechanism by which HSV-1 and P. aeruginosa can directly traffic to HGs and establish infection.

HSV-1 elicits a robust inflammatory response within ocular glands subsequent to HSV-1 keratitis.Ocular glands are included in the mucosal immune system of the human host and are considered eye-associated lymphoid tissue (2). To determine if immune cells populate murine ocular glands during an uninfected state, glandular tissue was harvested and assessed for immune cells by histology and flow cytometry (Fig. 2). Hematoxylin and eosin (H&E) staining of ocular gland tissue sections revealed areas of inflammation in infected but not uninfected HGs and ELGs (Fig. 2). Within uninfected glands, myeloid-derived cells (CD45+ CD11b+) and T lymphocytes (CD45+ CD3+ CD4+ and CD45+ CD3+ CD8+), but not B lymphocytes (CD45+ CD19+), were present, with significantly more CD11b+ and T (CD4+ and CD8+) cells found in ELGs (Fig. 2B) than in HGs, as measured by flow cytometry (Fig. 3A). To determine whether HSV-1 infection of ocular glands altered the local immune cell profile, CD-1 mice were corneally scarified and topically infected with HSV-1, and glands were collected at days 3, 5 and 7 p.i. In both HGs (Fig. 3A) and ELGs (Fig. 3B), a significant increase in the number of CD4+ T cells, CD8+ T cells, myeloid-derived cells, and B cells occurred by day 7 p.i.

To further characterize the immune profile of HGs and ELGs subsequent to HSV-1 corneal infection, the glands were surveyed for select cytokines and chemokines, comparing uninfected to infected tissue. The results showed that interleukin 4 (IL-4), IL-6, and IL-22 were elevated in ELGs but not HGs at day 7 p.i. (Fig. 4). Expression of gamma interferon (IFN-γ) was found to be elevated in both glands at day 7 p.i., in addition to chemokines CCL2, CCL5, and CXCL10 (Fig. 4). We interpret these results to suggest that a localized immune response within the ocular glands occurs subsequent to corneal HSV-1 infection, of which soluble factors, including chemokines and cytokines, may contribute to the immune milieu found within the tear film of the eye.

HSV-1 keratitis patients have been reported to experience decreased tear production during periods of inactive disease, suggesting prolonged dysfunction of processes involved in tear production (30, 31). Changes in the eicosanoid profile of tears have been recently implicated in aging and the dysfunction of Meibomian glands, accessory ocular glands that line the eyelids and make a significant contribution to the lipid portion of the tear film (32). Eicosanoids are signaling lipids capable of mediating both pro- and anti-inflammatory processes (33). We assessed HGs and ELGs for 157 eicosanoid metabolites at 30 days p.i. to determine whether ocular glands had an altered eicosanoid profile following the resolution of HSV-1 keratitis that occurs between 7 and 12 days p.i. (34). HGs contained decreased levels of 15-hydroxyeicosatrienoic acid (15-HETrE) and 8-HETrE at 30 days p.i. (Fig. 5A). 15-HETrE has been shown to inhibit the formation of proinflammatory LtB4 produced by neutrophils (35). ELGs had decreased levels of TxB2 but elevated levels of 9(10)-epoxy-12Z-octadecanoic acid [9(10)-EpOME], 9-oxoODE, and 13-oxoODE at 30 days p.i. (Fig. 5B). 9(10)-EpOME is a leukotoxin that is produced by inflammatory cells, including neutrophils and macrophages (36). These results suggest that infection of ocular glands secondary to microbial keratitis promotes an inflammatory eicosanoid profile well after the resolution of infection, which may be indicative of abnormal glandular function.

Removal of HGs increases the susceptibility of the cornea to HSV-1 infection.The above data suggest that HGs and ELGs are susceptible to HSV-1 infection secondary to corneal involvement and generate a dynamic, localized immune response. To begin to determine the role of these glands in corneal resistance to infection, surgery was performed in which HGs were removed from anesthetized CD-1 mice (50). Following a healing period of 21 days, corneas were assessed for changes in morphology by spectral domain-optical coherence tomography (SD-OCT) prior to infection. SD-OCT collects information on the light scatter of the cornea in order to construct a depth-based image that can be used to identify pathology and measure corneal thickness. Removal of HGs (HG–) did not alter corneal thickness (Fig. 6A and B) and did not result in a corneal epithelium defect, as measured by the inability of 1% fluorescein to enter the stroma in comparison to that in mice who received mock surgery (HG+ mice) (data not shown).

Ocular glands play a role in corneal resistance to HSV-1 keratitis. CD-1 mice received either harderian gland adenectomies (HG–) or mock surgery (HG+), rested for 21 days, and then were infected via addition of 150 to 250 PFU of HSV-1 to scarified corneas. (A and B) At 21 days following surgery and 5 days p.i., spectral domain-optical coherence tomography measurements of corneal thickness were taken. (C) At 7 days p.i., corneas from mice without HGs (HG–) and with HGs (HG+) were collected and assessed for viral content via plaque assay. (D) Infiltration of immune cells into the corneas of HG+ and HG– mice was assessed using flow cytometry at day 5 p.i., and cells were identified with markers CD45, CD11b, Ly6G, and Ly6C. Results represent two independent experiments of 2 to 5 mice per group/experiment. Statistics: Mann-Whitney rank sum test, P < 0.05 (*) and P < 0.01 (**).

Mice with and without HGs were then HSV-1 infected to determine if corneal resistance to infection was hindered by the absence of HGs. Removal of HGs increased the susceptibility of the cornea to HSV-1, as indicated by a significant elevation in infectious virus recovered from corneas at 7 days p.i. compared to that in mice who received mock surgery (Fig. 6C). This increase in the cornea’s susceptibility to HSV-1 was associated with decreased corneal edema (Fig. 6A and B) and decreased infiltration of innate immune cells (Fig. 6D) at 5 days p.i. Next, we used a 32-plex bead-based protein assay to determine whether the expression of inflammatory mediators both known and unknown to ocular HSV-1 had altered expression in this model (16). Surprisingly, no change was found in analyte expression between the corneas of mice with and without HGs at 5 days p.i. (Table 1). This may suggest that the majority of cytokines produced in the cornea 5 days p.i. are supplied by residential cells and not by infiltrating immune cells, whose numbers differ significantly between mice with and without HGs (Fig. 6D). This may also suggest that cytokine production within the cornea is independent of HG regulation or that some other ocular glands are compensating for HG removal. Collectively, these results implicate a role for HGs in promoting resistance to HSV-1 infection.

DISCUSSION

The involvement of ocular glands during corneal infection and their susceptibility to infection is an understudied area in microbial pathogenesis of the cornea. We were able to collect HSV-1 and P. aeruginosa from ocular glands subsequent to corneal infection. While we largely utilized HSV-1 for this study, the potential susceptibility of glands to a broader spectrum of pathogens should be appreciated. Cases of viral, bacterial, and fungal dacryoadenitis or inflammation of the lacrimal gland are seldom documented in humans, perhaps because treatment of the cornea may also serve to address simultaneous infection of ocular glands (22, 37, 38). While this study brings light to an unexplored area of ocular infection, further study is required to address the implications of glandular involvement on infection outcomes.

While HSV-1 replicates readily in epithelium, it is considered a neurotropic virus known to cause nerve degeneration and loss of sensation during corneal infection (39). Loss of corneal sensation results in a decreased blink reflex, increased corneal desiccation, and increased corneal inflammation (40). Ocular gland secretion is a process heavily regulated by innervating nerves and peripheral nerve signaling from the cornea (7). Ocular gland function may be subject to dysregulation during infection through loss of signaling from corneal nerves or damage to glandular nerves. Preventing corneal desiccation by tarsorrhaphy has been shown to significantly reduce inflammation during experimental corneal HSV-1 infection, despite unchanged damage to corneal nerves, suggesting that ocular surface moisture is an important determinant of corneal inflammation (40). Ocular surface moisture is provided by ocular glands, and decreased glandular function may result in a state of corneal desiccation and worsened inflammation. Tear production is decreased in ocular HSV-1 patients, even when corneal disease is asymptomatic (30, 31). The damage to ocular glands by previous ocular HSV-1 outbreaks may contribute to a chronic state of ocular gland dysfunction during otherwise asymptomatic periods of time.

This study describes changes in the immune profile of ocular glands during HSV-1 infection, including an increase in immune cells and proinflammatory mediators. In other disease models, such as age-related dry eye disease, increased numbers of immune cells within ocular glands have been reported to impact glandular function (4). CD4+ T cells within ocular glands are thought to contribute to dry eye disease pathology through production of proinflammatory IFN-γ and IL-17 cytokines (4). In our study, both CD4+ and CD8+ T cells were found to be elevated in ocular glands following HSV-1 infection, which may influence glandular processes. Protein expression of IL-4, IL-6, IL-22, IFN-γ, CCL2, CCL5, and CXCL10 was quantified, and expression of each analyte was found to be elevated in one or both infected ocular glands. While these factors are part of the glandular immune response, they may also regulate corneal immunity upon secretion onto the ocular surface. Furthermore, these inflammatory mediators are elevated in the tears of patients with dysfunctional tear production, suggesting that these factors may be useful biomarkers for ocular gland infection leading to dysregulation (41–43).

To determine the significance of ocular glands to corneal susceptibility to HSV-1, we utilized a previously established method for the removal of HGs (42). Removal of HGs alone resulted in no appreciable impact on corneal morphology, as determined by SD-OCT at 21 days postsurgery. While a limitation of our study is the removal of HGs and no other ocular glands, infection of HG adenectomized mice did result in decreased corneal edema, decreased inflammatory cell infiltration, and increased viral load within corneas. It has been shown that typical corneal HSV-1 infection results in corneal edema and infiltration of inflammatory immune cells (20). With the observations reported herein, we interpret these results to suggest that factors and cells within HGs contribute to resistance of the cornea to HSV-1 infection. Corneal epithelial cells have been shown to produce proinflammatory mediators in response to pathogens in vitro, where their function is independent of ocular gland secretions (44, 45). We speculate that the same proinflammatory mediators produced in ocular glands may contribute to and amplify corneal resistance upon ocular surface secretion. This study indicates that cells residing in HGs serve as a source of CXCL10 at 7 days p.i. A previous study found that depletion of CXCL10 during HSV-1 ocular infection increased the viral load measured in the corneas (46). We propose that elimination of HGs and the CXCL10 they provide contributes to a similar phenotype in our study, characterized by increased viral load and decreased inflammatory cell infiltration. Overall, this study suggests that ocular glands are susceptible to infection secondary to infectious keratitis, host a robust immune response, and contribute to corneal resistance to HSV-1. The mechanisms by which this occurs require further study.

In a paper submitted at the time of the submission of the present paper and recently published, another group reported that HSV-1 infected the ELG following corneal infection and that this infection led to a loss in tear production initially noted at day 7 p.i. (47). In addition, glandular pathology associated with CD4+ and CD8+ T cell infiltration and expression of IFN-γ was described and was correlative to the severity of HSV-1 keratitis. Together, these two independent reports demonstrate the susceptibility of extraorbital ocular glands to HSV-1 infection following cornea exposure to virus, tissue pathology, and inflammation, including cytokine production by resident or infiltrating cells that results in the loss of tear production consistent with what has been reported in the human patient (30, 31).

MATERIALS AND METHODS

Mouse models of HSV-1 and P. aeruginosa infection.Male and female outbred CD-1 mice (8 to 12 weeks of age; Charles River, Wilmington, MA) were housed in a specific-pathogen-free animal facility at the Dean McGee Eye Institute. This study was approved by the OUHSC animal care and use committee (protocol no. 16-014 for HSV-1 and 18-009 for P. aeruginosa) and performed in adherence to the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research. Mice were anesthetized immediately prior to infection with an intraperitoneal injection of ketamine (100 mg/kg) and xylazine (6.6 mg/kg). For HSV-1, mice were infected via scarification of the corneal epithelium using a 25-gauge needle in a crosshatch pattern with 20 vertical and 20 horizontal scratches. HSV-1 (strain McKrae, 150 to 250 PFU) was added to the ocular surface in a 3-μl volume of phosphate-buffered saline (PBS). For P. aeruginosa infection, mice were anesthetized at the time of infection as described above and infected via scarification of the corneal epithelium with an 18-gauge needle 3 times. Approximately 4 × 107 CFU of P. aeruginosa strain PAO1 in 10 μl of brain heart infusion (BHI) medium was applied to the corneal surface and allowed to sit for 5 min. Excess inocula was wicked away using a Kimwipe. In both HSV-1 and P. aeruginosa infection models, uninfected mice received no scarification and no inoculum. At the time of tissue harvest, mice were anesthetized and euthanized by cardiac perfusion with10 to 20 ml of PBS. After euthanasia, the numbers of P. aeruginosa CFU in eyes, HGs, and ELGs were quantified by track dilution on BHI agar, as described previously (48, 49). Colonies were confirmed as Pseudomonas on Pseudomonas isolation agar (Sigma).

Harderian gland adenectomy.Following anesthesia, the skin and fur surrounding each eye were carefully cleansed with 70% ethanol, avoiding direct eye contact. The conjunctiva located at the medial canthus was clipped to create an ∼2-mm opening. Small forceps were inserted into the opening, and the HG was grasped against the inner orbit. Connective tissue attaching the HG to the orbital space was trimmed using scissors. The HG was then removed by grasping the gland and extracting it through the 2-mm opening in the conjunctiva. Pressure was then applied until bleeding discontinued. Mice that received mock surgery had their conjunctiva clipped at the medial canthus. During clipping, the forceps briefly entered the retroorbital space but did not disturb the contents of the space. Immediately following surgery or mock surgery, mice received topical bacitracin zinc antibiotic applied to their ocular surface and lid area, as well as an intraperitoneal injection of 0.01 mg/kg buprenorphine. Mice received hydrogel diet supplement as well as topical antibiotics and buprenorphine twice daily for 3 days. The mice were allowed to heal for 21 days postsurgery prior to infection with HSV-1.

Viral titer.Following corneal infection, animals were euthanized at days 3, 5 and 7 postinfection (p.i.) and the corneas, ELGs, HGs, and TGs were collected and frozen at −80°C until plaque assays were performed as previously described (21).

Histology.Following corneal infection, animals were euthanized at 7 days p.i. HGs and ELGs were removed following cardiac perfusion with 10 ml of PBS. Tissues were fixed in 4% paraformaldehyde at room temperature for 5 h. Paraffin embedding, sectioning, and H&E staining were performed by the Dean McGee Eye Institute Imaging Core staff. Images were obtained using a Nikon E800 epifluorescent microscope with MetaVue image capture software.

Flow cytometry.HGs, ELGs, and mandibular lymph nodes (MLNs) were collected at days 3, 5, and 7 p.i. HGs and ELGs were digested in 1.0 ml of 2.0 mg/ml type 1 collagenase (Sigma Aldrich, St. Louis, MO) in RPMI 1640 medium containing 10% fetal bovine serum (FBS) and antimycotic/antibiotic solution (complete medium). Tissues were incubated in a 37°C water bath for 60 min and then triturated every 15 min for up to an additional 1.5 h. The resulting single-cell suspension was filtered through a 40-μm mesh, centrifuged at 300 × g for 5 min, and decanted, and the pellet was resuspended in 2.0 ml of complete medium. MLNs were macerated into a single-cell suspension in 3.5 ml of complete medium. Single-cell suspensions were then incubated in 1 to 2 μl Fc block CD16/CD32 (Thermo Fisher Scientific, Waltham, MA) diluted in 1% bovine serum albumin (BSA) in 1× PBS for 15 min and labeled with a combination of 1 μl each of CD45 efluor450, CD19 phycoerythrin (PE), B220 allophycocyanin (APC)-Cy7, EpCAM efluor450, CD4 APC-Cy7, CD8 PE, CD11b fluorescein isothiocyanate (FITC) and PE-Cy7, Ly6-G APC, Ly6-C PE, and CD11c PE-Cy7 (Thermo Fisher Scientific) diluted in 1% BSA in 1× PBS for 30 min. Samples were then washed twice by adding 1 ml of 1% BSA in 1× PBS, centrifuging the mixture for 5 min at 300 × g, and decanting the supernatant. Samples were then fixed in 1 ml of 1% paraformaldehyde overnight and resuspended in 1 ml of 1% BSA in 1× PBS to be analyzed on a MACSQuant 196 flow cytometer (Miltenyi Biotech). Gating strategies were consistent with what has previously been described (21).

Ocular surface content bidirectional flow assay.Following anesthesia, 3 μl of 1% Evans blue dye or PBS was topically applied to the scarified corneas of the mice. After 4 h, the animals were euthanized as described above and the HGs were collected. HGs were placed on white paper and photographed with a Nikon D100 camera and a Nikon AF Micro-Nikkor 60-mm lens. Adobe Photoshop CS5 was used to enhance brightness identically for each sample.

SD-OCT of cornea.Following HG adenectomy (or mock) surgery and a healing period, the corneas of mice were imaged before and after (5 days p.i.) infection using a Bioptigen spectral domain-optical coherence tomography (SD-OCT) system (Leica Microsystems). Mice were anesthetized and positioned so that the Purkinje reflection was located in the center of the cornea. Collected images were quantified for corneal thickness using calipers built into the Bioptigen software.

Real-time PCR.RNA was isolated from tissue homogenized in TRIzol reagent (Invitrogen), according to the manufacturer’s instructions. Five hundred nanograms of cDNA was generated from each RNA sample by using the iScript reverse transcription supermix (Bio-Rad). Real-time PCR (RT-PCR) was performed with the iTaq Universal SYBR green supermix (Bio-Rad) and primers (Denovo Biotechnology) for the HSV-1 thymidine kinase (TK) gene (forward, 5′-ATACCGACGATCTGCGACCT-3′; reverse, 5′-TTATTGCCGTCATAGCGCGG-3′) and the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene (forward, 5′-AGAATTGACAAACGGGACCT-3′; reverse, 5′-GGAGGAGCAGAGAGCTTGAC-3′) using a CFX96 thermocycler (Bio-Rad). Relative expression of the HSV-1 TK gene was normalized to GAPDH gene expression. Samples with no HSV-1 TK signal (uninfected sample) were assigned a threshold cycle (CT) value of 40. The ΔΔCT was calculated as previously described (21).

Measuring epithelial defect.One percent fluorescein was placed onto the ocular surface of anesthetized mice and mechanically dispersed. Mice were then imaged using a Micron IV microscope with a cobalt blue filter and Discover software. Fluorescein staining of corneas indicates an epithelial defect based on fluorescein’s ability to stain stromal, but not epithelial, cells. Images were quantified in ImageJ by converting images to gray scale and measuring fluorescein intensity (white) above background (black).

Eicosanoid analysis.At 30 days p.i., HGs and ELGs were removed, flash frozen with liquid nitrogen, and stored at −80°C before shipment to the Lipid MAPS Consortium lipidomics service core at the University of California San Diego (http://www.ucsd-lipidmaps.org/). The samples were assessed for eicosanoids included in the comprehensive eicosanoid panel, consisting of 157 eicosanoid metabolites, using liquid chromatography-tandem mass spectrometry.

Statistical analysis.GraphPad Prism 5 was used to statistically analyze data sets presented as the mean ± standard error of the mean. The Mann-Whitney rank sum test or Welch’s t test was used to determine a significant (P ≤ 0.05) difference between two groups. Kruskal-Wallis one-way analyses of variance (ANOVA) with Dunn’s multiple-comparison test was used to determine significant (P ≤ 0.05) differences between multiple groups.

ACKNOWLEDGMENTS

This work was supported by NIH R01 EY021238, NEI core grant P30 EY021725, and an unrestricted grant from Research to Prevent Blindness.

We thank the staff of the Dean McGee Eye Institute animal facility for their efforts in maintaining and monitoring our mice. We thank Linda Boone at the Dean McGee Eye Institute’s Imaging Core for her assistance in preparing histological slides. We thank Derek Royer, Meghan Carr, Phillip Coburn, Roger Astley, and Renee Sallack for their technical assistance.